Which enzyme introduces negative supercoils into bacterial DNA?

The enzyme that introduces negative supercoils into bacterial DNA is DNA gyrase.

DNA gyrase is a type II topoisomerase. Using energy from ATP, it temporarily cuts both strands of a DNA segment, passes another segment through the break, and reseals the DNA. This strand-passage reaction changes the DNA’s linking number and actively introduces negative supercoils, making the bacterial chromosome more compact and easier to open for replication and transcription.

Gyrase also removes positive supercoils that build up ahead of RNA polymerase or a replication fork. That role is especially important because unwinding the double helix creates torsional strain in the DNA ahead of the molecular machinery. Topoisomerase IV can also relax positive supercoils and separate daughter chromosomes, but it is not the classic enzyme that actively creates negative supercoiling.

The distractors perform different jobs: DNA polymerase synthesizes DNA, DNA ligase seals breaks, and DNA helicase separates the two strands. DNA gyrase is also a major antibiotic target; fluoroquinolones such as ciprofloxacin interfere with gyrase and related topoisomerase activity.